Our objective is to define the functions of Kell and XK, the two proteins of the Kell blood group system. On red cells Kell and XK are linked by a single disulfide bond. Kell is a 93 kDa type II membrane glycoprotein that preferentially activates endothelin-3. XK is predicted to be a 50.9 kDa protein that has the structural characteristics of a transporter but its function is not known. However, absence of XK, the McLeod phenotype, is associated with red cell acanthocytosis and late onset forms of neuromuscular dysfunctions. To meet our objective we have the following specific aims. (1) To determine the biochemical relationship and cellular locations of Keg and XK in different mouse tissues. In RBCs Kell and XK exist predominantly as a heterodimer. However this may not be the case in nonerythroid tissues where Kell and XK may exist separately. Although Kell is an ectoenzyme its optimal pH for enzyme activity is acidic, raising the possibility that Kell may also have an intracellular location and function. We will therefore determine the cellular locations and possible co-localization of Kell and XK proteins in mouse tissues. (2) To determine the transport functions of XK. Transport functions will be studied in mouse red cells and in transfected Xenopus oocytes. Since on red cells XK is linked to Kell, we postulate that the transport function of XK is modulated by endothelins. We will therefore compare the transport of various solutes in wild-type and Xk-/- red cells and determine if transport is affected by endothelins. We will also express XK, and XK/Kell in Xenopus oocytes and measure membrane conductance by the two-electrode voltage clamp procedure. (3) To determine, utilizing mice with targeted disruption of Kel and Xk, if Kell and XK proteins have complementary functions. The phenotype of double-knockout mice, Kel-/-, Xk-/-, will be compared to that of mice with disruption of only Xk. Emphasis will be placed on differences in the pathology of skeletal muscle and brain, the reproductive system and functions known to be affected by the endothelins. (4). To determine the physiological function of Kell. Is Kell activated in microenvironments that foster low pH? We propose that Kell, as an ectoenzyme with an acidic pH optimum, functions in acidic microenvironments. Acidic microenvironments are known to occur in several situations, such as vascular injury and epidermal wounds. We will determine, utilizing wild-type and Kel-/- mice, whether Kell, as an endothelin-converting enzyme, actively participates in an acidic microenvironment to promote neovascularization and cell proliferation which are integral to wound healing. We anticipate that findings from our proposed studies will shed important insights into the functions of two important but poorly understood red cell membrane proteins.